Cloning using donor nuclei from differentiated fetal and adult cells

ABSTRACT

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 08/935,052,filed Sep. 22, 1997, which is a divisional of U.S. Ser. No. 08/781,752,filed Jan. 10, 1997, and which are incorporated herein in their entiretyby reference.

FIELD OF THE INVENTION

[0002] The present invention relates to cloning procedures in which cellnuclei derived from differentiated fetal or adult, mammalian cells aretransplanted into enucleated mammalian oocytes of the same species asthe donor nuclei. The nuclei are reprogrammed to direct the developmentof cloned embryos, which can then be transferred into recipient femalesto produce fetuses and offspring, or used to produce cultured inner cellmass cells (CICM). The cloned embryos can also be combined withfertilized embryos to produce chimeric embryos, fetuses and/oroffspring.

BACKGROUND OF THE INVENTION

[0003] Methods for deriving embryonic stem (ES) cell lines in vitro fromearly preimplantation mouse embryos are well known. (See, e.g., Evans etal., Nature, 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA,78:7634-7638 (1981)). ES cells can be passaged in an undifferentiatedstate, provided that a feeder layer of fibroblast cells (Evans et al.,Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol.,121:1-9 (1987)) is present.

[0004] ES cells have been previously reported to possess numerousapplications. For example, it has been reported that ES cells can beused as an in vitro model for differentiation, especially for the studyof genes which are involved in the regulation of early development.Mouse ES cells can give rise to germline chimeras when introduced intopreimplantation mouse embryos, thus demonstrating their pluripotency(Bradley et al., Nature, 309:255-256 (1984)).

[0005] In view of their ability to transfer their genome to the nextgeneration, ES cells have potential utility for germline manipulation oflivestock animals by using ES cells with or without a desired geneticmodification. Moreover, in the case of livestock animals, e.g.,ungulates, nuclei from like preimplantation livestock embryos supportthe development of enucleated oocytes to term (Smith et al., Biol.Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod.,50:935-939 (1994)). This is in contrast to nuclei from mouse embryoswhich beyond the eight-cell stage after transfer reportedly do notsupport the development of enucleated oocytes (Cheong et al, Biol.Reprod., 48:958 (1993)). Therefore, ES cells from livestock animals arehighly desirable because they may provide a potential source oftotipotent donor nuclei, genetically manipulated or otherwise, fornuclear transfer procedures.

[0006] Some research groups have reported the isolation of purportedlypluripotent embryonic cell lines. For example, Notarianni et al., J.Reprod. Fert. Suppl., 43:255-260 (1991), reports the establishment ofpurportedly stable, pluripotent cell lines from pig and sheepblastocysts which exhibit some morphological and growth characteristicssimilar to that of cells in primary cultures of inner cell massesisolated immunosurgically from sheep blastocysts. Also, Notarianni etal., J. Reprod. Fert. Suppl., 41:51-56 (1990) discloses maintenance anddifferentiation in culture of putative pluripotential embryonic celllines from pig blastocysts. Gerfen et al., Anim. Biotech, 6(1):1-14(1995) discloses the isolation of embryonic cell lines from porcineblastocysts. These cells are stably maintained in mouse embryonicfibroblast feeder layers without the use of conditioned medium, andreportedly differentiate into several different cell types duringculture.

[0007] Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141(1992) reports cultured, bovine embryonic stem cell-like cell lineswhich survived three passages, but were lost after the fourth passage.Handyside et al., Roux's Arch. Dev. Biol., 196:185-190 (1987) disclosesculturing of immunosurgically isolated inner cell masses of sheepembryos under conditions which allow for the isolation of mouse ES celllines derived from mouse ICMs. Handyside et al. reports that under suchconditions, the sheep ICMs attach, spread, and develop areas of both EScell-like and endoderm-like cells, but that after prolonged culture onlyendoderm-like cells are evident.

[0008] Recently, Cherny et al., Theriogenology, 41:175 (1994) reportedpurportedly pluripotent bovine primordial germ cell-derived cell linesmaintained in long-term culture. These cells, after approximately sevendays in culture, produced ES-like colonies which stained positive foralkaline phosphatase (AP), exhibited the ability to form embryoidbodies, and spontaneously differentiated into at least two differentcell types. These cells also reportedly expressed mRNA for thetranscription factors OCT4, OCT6 and HES 1, a pattern of homeobox geneswhich is believed to be expressed by ES cells exclusively.

[0009] Also recently, Campbell et al., Nature, 380:64-68 (1996) reportedthe production of live lambs following nuclear transfer of culturedembryonic disc (ED) cells from day nine ovine embryos cultured underconditions which promote the isolation of ES cell lines in the mouse.The authors concluded that ED cells from day nine ovine embryos aretotipotent by nuclear transfer and that totipotency is maintained inculture.

[0010] Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454(1995), reported the isolation and characterization of purportedlypermanent cell lines from inner cell mass cells of bovine blastocysts.The authors isolated and cultured ICMs from 8 or 9 day bovineblastocysts under different conditions to determine which feeder cellsand culture media are most efficient in supporting the attachment andoutgrowth of bovine ICM cells. They concluded that the attachment andoutgrowth of cultured ICM cells is enhanced by the use of STO (mousefibroblast) feeder cells (instead of bovine uterus epithelial cells) andby the use of charcoal-stripped serum (rather than normal serum) tosupplement the culture medium. Van Stekelenburg et al reported, however,that their cell lines resembled epithelial cells more than pluripotentICM cells.

[0011] Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et al,WO 90/03432, published Apr. 5, 1990, and Wheeler et al, WO 94/26889,published Nov. 24, 1994, report the isolation, selection and propagationof animal stem cells which purportedly may be used to obtain transgenicanimals. Evans et al. also reported the derivation of purportedlypluripotent embryonic stem cells from porcine and bovine species whichassertedly are useful for the production of transgenic animals. Further,Wheeler et al, WO 94/26884, published Nov. 24, 1994, disclosed embryonicstem cells which are assertedly useful for the manufacture of chimericand transgenic ungulates.

[0012] Thus, based on the foregoing, it is evident that many groups haveattempted to produce ES cell lines, e.g., because of their potentialapplication in the production of cloned or transgenic embryos and innuclear transplantation.

[0013] The use of ungulate inner cell mass (ICM) cells for nucleartransplantation has also been reported. For example, Collas et al., Mol.Reprod. Dev., 38:264-267 (1994) discloses nuclear transplantation ofbovine ICMs by microinjection of the lysed donor cells into enucleatedmature oocytes. Collas et al. disclosed culturing of embryos in vitrofor seven days to produce fifteen blastocysts which, upon transferralinto bovine recipients, resulted in four pregnancies and two births.Also, Keefer et al., Biol. Reprod., 50:935-939 (1994), disclosed the useof bovine ICM cells as donor nuclei in nuclear transfer procedures, toproduce blastocysts which, upon transplantation into bovine recipients,resulted in several live offspring. Further, Sims et al., Proc. Natl.Acad. Sci., USA, 90:6143-6147 (1993), disclosed the production of calvesby transfer of nuclei from short-term in vitro cultured bovine ICM cellsinto enucleated mature oocytes.

[0014] The production of live lambs following nuclear transfer ofcultured embryonic disc cells has also been reported (Campbell et al.,Nature, 380:64-68 (1996)). Still further, the use of bovine pluripotentembryonic cells in nuclear transfer and the production of chimericfetuses has been reported (Stice et al., Biol. Reprod., 54:100-110(1996); Collas et al, Mol. Reprod. Dev., 38:264-267 (1994)). Collas etal demonstrated that granulosa cells (adult cells) could be used in abovine cloning procedure to produce embryos. However, there was nodemonstration of development past early embryonic stages (blastocyststage). Also, granulosa cells are not easily cultured and are onlyobtainable from females. Collas et al did not attempt to propagate thegranulosa cells in culture or try to genetically modify those cells.

[0015] While multiplications of genotypes are possible using embryoniccells as donors, there are problems with current methods. For example,by current methods, embryo cloning can only be done using a limitednumber of embryonic donor nuclei (less than 100), or with in vitro celllines. It is unknown whether the embryonic genome encodes a superiorgenotype until the cloned animal becomes an adult.

[0016] There also exist problems in the area of producing transgenicmammals. By current methods, heterologous DNA is introduced into eitherearly embryos or embryonic cell lines that differentiate into variouscell types in the fetus and eventually develop into a transgenic animal.However, many early embryos are required to produce one transgenicanimal and, thus, this procedure is very inefficient. Also, there is nosimple and efficient method of selecting for a transgenic embryo beforegoing through the time and expense of putting the embryos into surrogatefemales. In addition, gene targeting techniques cannot be easilyaccomplished with early embryo transgenic procedures.

[0017] Embryonic stem cells in mice have enabled researchers to selectfor transgenic cells and perform gene targeting. This allows moregenetic engineering than is possible with other transgenic techniques.However, embryonic stem cell lines and other embryonic cell lines mustbe maintained in an undifferentiated state that requires feeder layersand/or the addition of cytokines to media. Even if these precautions arefollowed, these cells often undergo spontaneous differentiation andcannot be used to produce transgenic offspring by currently availablemethods. Also, some embryonic cell lines have to be propagated in a waythat is not conducive to gene targeting procedures.

[0018] Therefore, notwithstanding what has previously been reported inthe literature, there exists a need for improved methods of cloningmammalian cells.

OBJECTS AND SUMMARY OF THE INVENTION

[0019] It is an object of the invention to provide novel and improvedmethods for producing cloned mammalian cells.

[0020] It is a more specific object of the invention to provide a novelmethod for cloning mammalian cells which involves transplantation of thenucleus of a differentiated mammalian cell into an enucleated oocyte ofthe same species.

[0021] It is another object of the invention to provide a method formultiplying adult mammals having proven genetic superiority or otherdesirable traits.

[0022] It is another object of the invention to provide an improvedmethod for producing genetically engineered or transgenic mammals (i.e.,embryos, fetuses, offspring).

[0023] It is a more specific object of the invention to provide a methodfor producing genetically engineered or transgenic mammals by which adesired gene is inserted, removed or modified in a differentiatedmammalian cell or cell nucleus prior to use of that differentiated cellor cell nucleus for formation of a NT unit.

[0024] It is another object of the invention to provide geneticallyengineered or transgenic mammals (i.e., embryos, fetuses, offspring)obtained by transplantation of the nucleus of a differentiated cell intoan enucleated oocyte of the same species as the differentiated cell.

[0025] It is another object of the invention to provide a novel methodfor producing mammalian CICM cells which involves transplantation of anucleus of a differentiated cell into an enucleated oocyte of the samespecies as the differentiated cell.

[0026] It is another object of the invention to provide CICM cellsproduced by transplantation of the nucleus of a differentiated mammaliancell into an enucleated oocyte of the same species as the differentiatedcell.

[0027] It is a more specific object of the invention to provide a methodfor producing human CICM cells which involves transplantation of nucleiof a human cell, e.g., a human adult cell, into an enucleated humanoocyte.

[0028] It is another object of the invention to use such CICM cells fortherapy or diagnosis.

[0029] It is a specific object of the invention to use such CICM cells,including human and ungulate CICM cells, for treatment or diagnosis ofany disease wherein cell, tissue or organ transplantation istherapeutically or diagnostically beneficial. The CICM cells may be usedwithin the same species or across species.

[0030] It is another object of the invention to use tissues derived fromNT embryos, fetuses or offspring, including human and ungulate tissues,for treatment or diagnosis of any disease wherein cell, tissue or organtransplantation is therapeutically or diagnostically beneficial. Thetissues may be used within the same species or across species.

[0031] It is another specific object of the invention to use the CICMcells produced according to the invention for the production ofdifferentiated cells, tissues or organs.

[0032] It is a more specific object of the invention to use the humanCICM cells produced according to the invention for the production ofdifferentiated human cells, tissues or organs.

[0033] It is another specific object of the invention to use the CICMcells produced according to the invention in vitro, e.g. for study ofcell differentiation and for assay purposes, e.g. for drug studies.

[0034] It is another object of the invention to provide improved methodsof transplantation therapy, comprising the usage of isogenic or syngeniccells, tissues or organs produced from the CICM cells produced accordingto the invention. Such therapies include by way of example treatment ofdiseases and injuries including Parkinson's, Huntington's, Alzheimer's,ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy,diabetes, liver diseases, heart disease, cartilage replacement, bums,vascular diseases, urinary tract diseases, as well as for the treatmentof immune defects, bone marrow transplantation, cancer, among otherdiseases.

[0035] It is another object of the invention to provide geneticallyengineered or transgenic CICM cells produced by inserting, removing ormodifying a desired gene in a differentiated mammalian cell or cellnucleus prior to use of that differentiated cell or cell nucleus forformation of a NT unit.

[0036] It is another object of the invention to use the transgenic orgenetically engineered CICM cells produced according to the inventionfor gene therapy, in particular for the treatment and/or prevention ofthe diseases and injuries identified, supra.

[0037] It is another object of the invention to use the CICM cellsproduced according to the invention or transgenic or geneticallyengineered CICM cells produced according to the invention as nucleardonors for nuclear transplantation.

[0038] Thus, in one aspect, the present invention provides a method forcloning a mammal (e.g., embryos, fetuses, offspring). The methodcomprises:

[0039] (i) inserting a desired differentiated mammalian cell or cellnucleus into an enucleated mammalian oocyte of the same species as thedifferentiated cell or cell nucleus, under conditions suitable for theformation of a nuclear transfer (NT) unit;

[0040] (ii) activating the resultant nuclear transfer unit;

[0041] (iii) culturing said activated nuclear transfer unit untilgreater than the 2-cell developmental stage; and

[0042] (iv) transferring said cultured NT unit to a host mammal suchthat the NT unit develops into a fetus.

[0043] The cells, tissues and/or organs of the fetus are advantageouslyused in the area of cell, tissue and/or organ transplantation.

[0044] The present invention also includes a method of cloning agenetically engineered or transgenic mammal, by which a desired gene isinserted, removed or modified in the differentiated mammalian cell orcell nucleus prior to insertion of the differentiated mammalian cell orcell nucleus into the enucleated oocyte.

[0045] Also provided by the present invention are mammals obtainedaccording to the above method, and offspring of those mammals.

[0046] The present invention is preferably used for cloning ungulates.

[0047] In another aspect, the present invention provides a method forproducing CICM cells. The method comprises:

[0048] (i) inserting a desired differentiated mammalian cell or cellnucleus into an enucleated mammalian oocyte of the same species as thedifferentiated cell or cell nucleus, under conditions suitable for theformation of a nuclear transfer (NT) unit;

[0049] (ii) activating the resultant nuclear transfer unit;

[0050] (iii) culturing said activated nuclear transfer unit untilgreater than the 2-cell developmental stage; and

[0051] (iv) culturing cells obtained from said cultured NT unit toobtain CICM cells.

[0052] The CICM cells are advantageously used in the area of cell,tissue and organ transplantation.

[0053] With the foregoing and other objects, advantages and features ofthe invention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides improved procedures for cloningmammals by nuclear transfer or nuclear transplantation. In the subjectapplication, nuclear transfer or nuclear transplantation or NT are usedinterchangeably.

[0055] According to the invention, cell nuclei derived fromdifferentiated fetal or adult, mammalian cells are transplanted intoenucleated mammalian oocytes of the same species as the donor nuclei.The nuclei are reprogrammed to direct the development of cloned embryos,which can then be transferred into recipient females to produce fetusesand offspring, or used to produce CICM cells. The cloned embryos canalso be combined with fertilized embryos to produce chimeric embryos,fetuses and/or offspring.

[0056] Prior art methods have used embryonic cell types in cloningprocedures. This includes work by Campbell et al (Nature, 380:64-68,1996) and Stice et al (Biol. Reprod., 54:100-110, 1996). In both ofthose studies, embryonic cell lines were derived from embryos of lessthan 10 days of gestation. In both studies, the cells were maintained ona feeder layer to prevent overt differentiation of the donor cell to beused in the cloning procedure. The present invention uses differentiatedcells.

[0057] It was unexpected that cloned embryos with differentiated donornuclei could develop to advanced embryonic and fetal stages. Thescientific dogma has been that only embryonic or undifferentiated celltypes could direct this type of development. It was unexpected that alarge number of cloned embryos could be produced from thesedifferentiated cell types. Also, the fact that new transgenic embryoniccell lines could be readily derived from transgenic cloned embryos wasunexpected.

[0058] Thus, according to the present invention, multiplication ofsuperior genotypes of mammals, including ungulates, is possible. Thiswill allow the multiplication of adult animals with proven geneticsuperiority or other desirable traits. Progress will be accelerated, forexample, in many important ungulate species. By the present invention,there are potentially billions of fetal or adult cells that can beharvested and used in the cloning procedure. This will potentiallyresult in many identical offspring in a short period.

[0059] The present invention also allows simplification of transgenicprocedures by working with a differentiated cell source that can beclonally propagated. This eliminates the need to maintain the cells inan undifferentiated state, thus, genetic modifications, both randomintegration and gene targeting, are more easily accomplished. Also bycombining nuclear transfer with the ability to modify and select forthese cells in vitro, this procedure is more efficient than previoustransgenic embryo techniques. According to the present invention, thesecells can be clonally propagated without cytokines, conditioned mediaand/or feeder layers, further simplifying and facilitating thetransgenic procedure. When transfected cells are used in cloningprocedures according to the invention, transgenic embryos are producedwhich can develop into fetuses and offspring. Also, these transgeniccloned embryos can be used to produce CICM cell lines or other embryoniccell lines. Therefore, the present invention eliminates the need toderive and maintain in vitro an undifferentiated cell line that isconducive to genetic engineering techniques.

[0060] The present invention can also be used to produce CICM cells,fetuses or offspring which can be used, for example, in cell, tissue andorgan transplantation. By taking a fetal or adult cell from an animaland using it in the cloning procedure a variety of cells, tissues andpossibly organs can be obtained from cloned fetuses as they developthrough organogenesis. Cells, tissues, and organs can be isolated fromcloned offspring as well. This process can provide a source of“materials” for many medical and veterinary therapies including cell andgene therapy. If the cells are transferred back into the animal in whichthe cells were derived, then immunological rejection is averted. Also,because many cell types can be isolated from these clones, othermethodologies such as hematopoietic chimerism can be used to avoidimmunological rejection among animals of the same species as well asbetween species.

[0061] Thus, in one aspect, the present invention provides a method forcloning a mammal. In general, the mammal will be produced by a nucleartransfer process comprising the following steps:

[0062] (i) obtaining desired differentiated mammalian cells to be usedas a source of donor nuclei;

[0063] (ii) obtaining oocytes from a mammal of the same species as thecells which are the source of donor nuclei;

[0064] (iii) enucleating said oocytes;

[0065] (iv) transferring the desired differentiated cell or cell nucleusinto the enucleated oocyte, e.g., by fusion or injection, to form NTunits;

[0066] (v) activating the resultant NT unit;

[0067] (vi) culturing said activated nuclear transfer unit until greaterthan the 2-cell developmental stage; and

[0068] (vii) transferring said cultured NT unit to a host mammal suchthat the NT unit develops into a fetus.

[0069] The present invention also includes a method of cloning agenetically engineered or transgenic mammal, by which a desired gene isinserted, removed or modified in the differentiated mammalian cell orcell nucleus prior to insertion of the differentiated mammalian cell orcell nucleus into the enucleated oocyte.

[0070] Also provided by the present invention are mammals obtainedaccording to the above method, and offspring of those mammals. Thepresent invention is preferably used for cloning ungulates.

[0071] The present invention further provides for the use of NT fetusesand NT and chimeric offspring in the area of cell, tissue and organtransplantation.

[0072] In another aspect, the present invention provides a method forproducing CICM cells. The method comprises:

[0073] (i) inserting a desired differentiated mammalian cell or cellnucleus into an enucleated mammalian oocyte of the same species as thedifferentiated cell or cell nucleus, under conditions suitable for theformation of a nuclear transfer (NT) unit;

[0074] (ii) activating the resultant nuclear transfer unit;

[0075] (iii) culturing said activated nuclear transfer unit untilgreater than the 2-cell developmental stage; and

[0076] (iv) culturing cells obtained from said cultured NT unit toobtain CICM cells.

[0077] The CICM cells are advantageously used in the area of cell,tissue and organ transplantation, or in the production of fetuses oroffspring, including transgenic fetuses or offspring.

[0078] Preferably, the NT units will be cultured to a size of at least 2to 400 cells, preferably 4 to 128 cells, and most preferably to a sizeof at least about 50 cells.

[0079] Nuclear transfer techniques or nuclear transplantation techniquesare known in the literature and are described in many of the referencescited in the Background of the Invention. See, in particular, Campbellet al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev.,38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Simset al, Proc. Natl. Acad. Sci, USA, 90:6143-6147 (1993); WO 94/26884; WO94/24274, and WO 90/03432, which are incorporated by reference in theirentirety herein. Also, U.S. Pat. Nos. 4,944,384 and 5,057,420 describeprocedures for bovine nuclear transplantation.

[0080] Differentiated mammalian cells are those cells which are past theearly embryonic stage. More particularly, the differentiated cells arethose from at least past the embryonic disc stage (day 10 of bovineembryogenesis). The differentiated cells may be derived from ectoderm,mesoderm or endoderm.

[0081] Mammalian cells, including human cells, may be obtained by wellknown methods. Mammalian cells useful in the present invention include,by way of example, epithelial cells, neural cells, epidermal cells,keratinocytes, hematopoietic cells, melanocytes, chondrocytes,lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes,mononuclear cells, fibroblasts, cardiac muscle cells, and other musclecells, etc. Moreover, the mammalian cells used for nuclear transfer maybe obtained from different organs, e.g., skin, lung, pancreas, liver,stomach, intestine, heart, reproductive organs, bladder, kidney, urethraand other urinary organs, etc. These are just examples of suitable donorcells. Suitable donor cells, i.e., cells useful in the subjectinvention, may be obtained from any cell or organ of the body. Thisincludes all somatic or germ cells.

[0082] Fibroblast cells are an ideal cell type because they can beobtained from developing fetuses and adult animals in large quantities.Fibroblast cells are differentiated somewhat and, thus, were previouslyconsidered a poor cell type to use in cloning procedures. Importantly,these cells can be easily propagated in vitro with a rapid doubling timeand can be clonally propagated for use in gene targeting procedures.Again the present invention is novel because differentiated cell typesare used. The present invention is advantageous because the cells can beeasily propagated, genetically modified and selected in vitro.

[0083] Suitable mammalian sources for oocytes include sheep, cows, pigs,horses, rabbits, guinea pigs, mice, hamsters, rats, primates, etc.Preferably, the oocytes will be obtained from ungulates, and mostpreferably bovine.

[0084] Methods for isolation of oocytes are well known in the art.Essentially, this will comprise isolating oocytes from the ovaries orreproductive tract of a mammal, e.g., a bovine. A readily availablesource of bovine oocytes is slaughterhouse materials.

[0085] For the successful use of techniques such as genetic engineering,nuclear transfer and cloning, oocytes must generally be matured in vitrobefore these cells may be used as recipient cells for nuclear transfer,and before they can be fertilized by the sperm cell to develop into anembryo. This process generally requires collecting immature (prophase I)oocytes from mammalian ovaries, e.g., bovine ovaries obtained at aslaughterhouse, and maturing the oocytes in a maturation medium prior tofertilization or enucleation until the oocyte attains the metaphase IIstage, which in the case of bovine oocytes generally occurs about 18-24hours post-aspiration. For purposes of the present invention, thisperiod of time is known as the “maturation period.” As used herein forcalculation of time periods, “aspiration” refers to aspiration of theimmature oocyte from ovarian follicles.

[0086] Additionally, metaphase II stage oocytes, which have been maturedin vivo have been successfully used in nuclear transfer techniques.Essentially, mature metaphase II oocytes are collected surgically fromeither non-superovulated or superovulated cows or heifers 35 to 48 hourspast the onset of estrus or past the injection of human chorionicgonadotropin (hCG) or similar hormone.

[0087] The stage of maturation of the oocyte at enucleation and nucleartransfer has been reported to be significant to the success of NTmethods. (See e.g., Prather et al., Differentiation, 48, 1-8, 1991). Ingeneral, successful mammalian embryo cloning practices use the metaphaseII stage oocyte as the recipient oocyte because at this stage it isbelieved that the oocyte can be or is sufficiently “activated” to treatthe introduced nucleus as it does a fertilizing sperm. In domesticanimals, and especially cattle, the oocyte activation period generallyranges from about 16-52 hours, preferably about 28-42 hourspost-aspiration. ,

[0088] For example, immature oocytes may be washed in HEPES bufferedhamster embryo culture medium (HECM) as described in Seshagine et al.,Biol. Reprod., 40, 544-606, 1989, and then placed into drops ofmaturation medium consisting of 50 microliters of tissue culture medium(TCM) 199 containing 10% fetal calf serum which contains appropriategonadotropins such as luteinizing hormone (LH) and follicle stimulatinghormone (FSH), and estradiol under a layer of lightweight paraffin orsilicon at 39° C.

[0089] After a fixed time maturation period, which ranges from about 10to 40 hours, and preferably about 16-18 hours, the oocytes will beenucleated. Prior to enucleation the oocytes will preferably be removedand placed in HECM containing 1 milligram per milliliter ofhyaluronidase prior to removal of cumulus cells. This may be effected byrepeated pipetting through very fine bore pipettes or by vortexingbriefly. The stripped oocytes are then screened for polar bodies, andthe selected metaphase II oocytes, as determined by the presence ofpolar bodies, are then used for nuclear transfer. Enucleation follows.

[0090] Enucleation may be effected by known methods, such as describedin U.S. Pat. No. 4,994,384 which is incorporated by reference herein.For example, metaphase II oocytes are either placed in HECM, optionallycontaining 7.5 micrograms per milliliter cytochalasin B, for immediateenucleation, or may be placed in a suitable medium, for example anembryo culture medium such as CR1 aa, plus 10% estrus cow serum, andthen enucleated later, preferably not more than 24 hours later, and morepreferably 16-18 hours later.

[0091] Enucleation may be accomplished microsurgically using amicropipette to remove the polar body and the adjacent cytoplasm. Theoocytes may then be screened to identify those of which have beensuccessfully enucleated. This screening may be effected by staining theoocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, andthen viewing the oocytes under ultraviolet irradiation for less than 10seconds. The oocytes that have been successfully enucleated can then beplaced in a suitable culture medium, e.g., CR1aa plus 10% serum.

[0092] In the present invention, the recipient oocytes will preferablybe enucleated at a time ranging from about 10 hours to about 40 hoursafter the initiation of in vitro maturation, more preferably from about16 hours to about 24 hours after initiation of in vitro maturation, andmost preferably about 16-18 hours after initiation of in vitromaturation.

[0093] A single mammalian cell of the same species as the enucleatedoocyte will then be transferred into the perivitelline space of theenucleated oocyte used to produce the NT unit. The mammalian cell andthe enucleated oocyte will be used to produce NT units according tomethods known in the art. For example, the cells may be fused byelectrofusion. Electrofulsion is accomplished by providing a pulse ofelectricity that is sufficient to cause a transient breakdown of theplasma membrane.

[0094] This breakdown of the plasma membrane is very short because themembrane reforms rapidly. Thus, if two adjacent membranes are induced tobreakdown and upon reformation the lipid bilayers intermingle, smallchannels will open between the two cells. Due to the thermodynamicinstability of such a small opening, it enlarges until the two cellsbecome one. Reference is made to U.S. Pat. No. 4,997,384 by Prather etal., (incorporated by reference in its entirety herein) for a furtherdiscussion of this process. A variety of electrofusion media can be usedincluding e.g., sucrose, mannitol, sorbitol and phosphate bufferedsolution. Fusion can also be accomplished using Sendai virus as afusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969).

[0095] Also, in some cases (e.g. with small donor nuclei) it may bepreferable to inject the nucleus directly into the oocyte rather thanusing electroporation fusion. Such techniques are disclosed in Collasand Barnes, Mol. Reprod. Dev., 38:264-267 (1994), incorporated byreference in its entirety herein.

[0096] Preferably, the mammalian cell and oocyte are electrofused in a500 μm chamber by application of an electrical pulse of 90-120V forabout 15 μsec, about 24 hours after initiation of oocyte maturation.After fusion, the resultant fused NT units are then placed in a suitablemedium until activation, e.g., CR1aa medium. Typically activation willbe effected shortly thereafter, typically less than 24 hours later, andpreferably about 4-9 hours later.

[0097] The NT unit may be activated by known methods. Such methodsinclude, e.g., culturing the NT unit at sub-physiological temperature,in essence by applying a cold, or actually cool temperature shock to theNT unit. This may be most conveniently done by culturing the NT unit atroom temperature, which is cold relative to the physiologicaltemperature conditions to which embryos are normally exposed.

[0098] Alternatively, activation may be achieved by application of knownactivation agents. For example, penetration of oocytes by sperm duringfertilization has been shown to activate prefusion oocytes to yieldgreater numbers of viable pregnancies and multiple genetically identicalcalves after nuclear transfer. Also, treatments such as electrical andchemical shock may be used to activate NT embryos after fusion. Suitableoocyte activation methods are the subject of U.S. Pat. No. 5,496,720, toSusko-Parrish et al., herein incorporated by reference in its entirety.

[0099] Additionally, activation may be effected by simultaneously orsequentially:

[0100] (i) increasing levels of divalent cations in the oocyte, and

[0101] (ii) reducing phosphorylation of cellular proteins in the oocyte.

[0102] This will generally be effected by introducing divalent cationsinto the oocyte cytoplasm, e.g., magnesium, strontium, barium orcalcium, e.g., in the form of an ionophore. Other methods of increasingdivalent cation levels include the use of electric shock, treatment withethanol and treatment with caged chelators.

[0103] Phosphorylation may be reduced by known methods, e.g., by theaddition of kinase inhibitors, e.g., serine-threonine kinase inhibitors,such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, andsphingosine.

[0104] Alternatively, phosphorylation of cellular proteins may beinhibited by introduction of a phosphatase into the oocyte, e.g.,phosphatase 2A and phosphatase 2B.

[0105] In one embodiment, NT activation is effected by briefly exposingthe fused NT unit to a TL-HEPES medium containing 5μM ionomycin and 1mg/ml BSA, followed by washing in TL-HEPES containing 30 mg/ml BSAwithin about 24 hours after fusion, and preferably about 4 to 9 hoursafter fusion.

[0106] The activated NT units may then be cultured in a suitable invitro culture medium until the generation of CICM cells and cellcolonies. Culture media suitable for culturing and maturation of embryosare well known in the art. Examples of known media, which may be usedfor bovine embryo culture and maintenance, include Ham's F-10+10% fetalcalf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calfserum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's PhosphateBuffered Saline (PBS), Eagle's and Whitten's media. One of the mostcommon media used for the collection and maturation of oocytes isTCM-199, and 1 to 20% serum supplement including fetal calf serum,newborn serum, estrual cow serum, lamb serum or steer serum. A preferredmaintenance medium includes TCM-199 with Earl salts, 10% fetal calfserum, 0.2 mM Na pyruvate and 50 μg/ml gentamicin sulphate. Any of theabove may also involve co-culture with a variety of cell types such asgranulosa cells, oviduct cells, BRL cells and uterine cells and STOcells.

[0107] Another maintenance medium is described in U.S. Pat. No.5,096,822 to Rosenkrans, Jr. et al., which is incorporated herein byreference. This embryo medium, named CR1, contains the nutritionalsubstances necessary to support an embryo.

[0108] CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mMto 10 mM, preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactate isL-lactate with a hemicalcium salt incorporated thereon. HemicalciumL-lactate is significant in that a single component satisfies two majorrequirements in the culture medium: (i) the calcium requirementnecessary for compaction and cytoskeleton arrangement; and (ii) thelactate requirement necessary for metabolism and electron transport.Hemicalcium L-lactate also serves as valuable mineral and energy sourcefor the medium necessary for viability of the embryos.

[0109] Advantageously, CR1 medium does not contain serum, such as fetalcalf serum, and does not require the use of a co-culture of animal cellsor other biological media, i.e., media comprising animal cells such asoviductal cells. Biological media can sometimes be disadvantageous inthat they may contain microorganisms or trace factors which may beharmful to the embryos and which are difficult to detect, characterizeand eliminate.

[0110] Examples of the main components in CR1 medium include hemicalciumL-lactate, sodium chloride, potassium chloride, sodium bicarbonate and aminor amount of fatty-acid free bovine serum albumin (Sigma A-6003).Additionally, a defined quantity of essential and non-essential aminoacids may be added to the medium. CR1 with amino acids is known by theabbreviation “CR1 aa.”

[0111] CR1 medium preferably contains the following components in thefollowing quantities: sodium chloride 114.7 mM potassium chloride 3.1 mMsodium bicarbonate 26.2 mM hemicalcium L-lactate 5 mM fatty-acid freeBSA 3 mg/ml

[0112] In one embodiment, the activated NT embryos unit are placed inCR1aa medium containing 1.9 mM DMAP for about 4 hours followed by a washin HECM and then cultured in CR1aa containing BSA.

[0113] For example, the activated NT units may be transferred to CR1aaculture medium containing 2.0 mM DMAP (Sigma) and cultured under ambientconditions, e.g., about 38.5° C., 5% CO₂ for a suitable time, e.g.,about 4 to 5 hours.

[0114] Afterward, the cultured NT unit or units are preferably washedand then placed in a suitable media, e.g., CR1aa medium containing 10%FCS and 6 mg/ml contained in well plates which preferably contain asuitable confluent feeder layer. Suitable feeder layers include, by wayof example, fibroblasts and epithelial cells, e.g., fibroblasts anduterine epithelial cells derived from ungulates, chicken fibroblasts,murine (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder celllines, and BRL cells.

[0115] In one embodiment, the feeder cells comprise mouse embryonicfibroblasts. Preparation of a suitable fibroblast feeder layer isdescribed in the example which follows and is well within the skill ofthe ordinary artisan.

[0116] The NT units are cultured on the feeder layer until the NT unitsreach a size suitable for transferring to a recipient female, or forobtaining cells which may be used to produce CICM cells or cellcolonies. Preferably, these NT units will be cultured until at leastabout 2 to 400 cells, more preferably about 4 to 128 cells, and mostpreferably at least about 50 cells. The culturing will be effected undersuitable conditions, i.e., about 38.5° C. and 5% CO₂, with the culturemedium changed in order to optimize growth typically about every 2-5days, preferably about every 3 days.

[0117] The methods for embryo transfer and recipient animal managementin the present invention are standard procedures used in the embryotransfer industry. Synchronous transfers are important for success ofthe present invention, i.e., the stage of the NT embryo is in synchronywith the estrus cycle of the recipient female. This advantage and how tomaintain recipients are reviewed in Siedel, G. E., Jr. (“Critical reviewof embryo transfer procedures with cattle” in Fertilization andEmbryonic Development in Vitro (1981) L. Mastroianni, Jr. and J. D.Biggers, ed., Plenum Press, New York, N.Y., page 323), the contents ofwhich are hereby incorporated by reference.

[0118] The present invention can also be used to clone geneticallyengineered or transgenic mammals. As explained above, the presentinvention is advantageous in that transgenic procedures can besimplified by working with a differentiated cell source that can beclonally propagated. In particular, the differentiated cells used fordonor nuclei have a desired gene inserted, removed or modified. Thosegenetically altered, differentiated cells are then used for nucleartransplantation with enucleated oocytes.

[0119] Any known method for inserting, deleting or modifying a desiredgene from a mammalian cell may be used for altering the differentiatedcell to be used as the nuclear donor. These procedures may remove all orpart of a gene, and the gene may be heterologous. Included is thetechnique of homologous recombination, which allows the insertion,deletion or modification of a gene or genes at a specific site or sitesin the cell genome.

[0120] The present invention can thus be used to provide adult mammalswith desired genotypes. Multiplication of adult ungulates with provengenetic superiority or other desirable traits is particularly useful,including transgenic or genetically engineered animals, and chimericanimals. Furthermore, cell and tissues from the NT fetus, includingtransgenic and/or chimeric fetuses, can be used in cell, tissue andorgan transplantation for the treatment of numerous diseases asdescribed below in connection with the use of CICM cells.

[0121] For production of CICM cells and cell lines, after NT units ofthe desired size are obtained, the cells are mechanically removed fromthe zone and are then used. This is preferably effected by taking theclump of cells which comprise the NT unit, which typically will containat least about 50 cells, washing such cells, and plating the cells ontoa feeder layer, e.g., irradiated fibroblast cells. Typically, the cellsused to obtain the stem cells or cell colonies will be obtained from theinner most portion of the cultured NT unit which is preferably at least50 cells in size. However, NT units of smaller or greater cell numbersas well as cells from other portions of the NT unit may also be used toobtain ES cells and cell colonies. The cells are maintained in thefeeder layer in a suitable growth medium, e.g., alpha MEM supplementedwith 10% FCS and 0.1 mM β-mercaptoethanol (Sigma) and L-glutamine. Thegrowth medium is changed as often as necessary to optimize growth, e.g.,about every 2-3 days.

[0122] This culturing process results in the formation of CICM cells orcell lines.

[0123] One skilled in the art can vary the culturing conditions asdesired to optimize growth of the particular CICM cells. Also,genetically engineered or transgenic mammalian CICM cells may beproduced according to the present invention. That is, the methodsdescribed above can be used to produce NT units in which a desired geneor genes have been introduced, or from which all or part of anendogenous gene or genes have been removed or modified. Thosegenetically engineered or transgenic NT units can then be used toproduce genetically engineered or transgenic CICM cells, including humancells.

[0124] The resultant CICM cells and cell lines, preferably human CICMcells and cell lines, have numerous therapeutic and diagnosticapplications. Most especially, such CICM cells may be used for celltransplantation therapies. Human CICM cells have application in thetreatment of numerous disease conditions. Human NT units per se may alsobe used in the treatment of disease conditions.

[0125] In this regard, it is known that mouse embryonic stem (ES) cellsare capable of differentiating into almost any cell type, e.g.,hematopoietic stem cells. Therefore, human CICM cells produced accordingto the invention should possess similar differentiation capacity. TheCICM cells according to the invention will be induced to differentiateto obtain the desired cell types according to known methods. Forexample, the subject human CICM cells may be induced to differentiateinto hematopoietic stem cells, muscle cells, cardiac muscle cells, livercells, cartilage cells, epithelial cells, urinary tract cells, etc., byculturing such cells in differentiation medium and under conditionswhich provide for cell differentiation. Medium and methods which resultin the differentiation of CICM cells are known in the art as aresuitable culturing conditions.

[0126] For example, Palacios et al, Proc. Natl. Acad. Sci., USA,92:7530-7537 (1995) teaches the production of hematopoietic stem cellsfrom an embryonic cell line by subjecting stem cells to an inductionprocedure comprising initially culturing aggregates of such cells in asuspension culture medium lacking retinoic acid followed by culturing inthe same medium containing retinoic acid, followed by transferral ofcell aggregates to a substrate which provides for cell attachment.

[0127] Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) isa review article which references numerous articles disclosing methodsfor in vitro differentiation of embryonic stem cells to produce variousdifferentiated cell types including hematopoietic cells, muscle, cardiacmuscle, nerve cells, among others.

[0128] Further, Bain et al, Dev. Biol., 168:342-357 (1995) teaches invitro differentiation of embryonic stem cells to produce neural cellswhich possess neuronal properties. These references are exemplary ofreported methods for obtaining differentiated cells from embryonic orstem cells. These references and in particular the disclosures thereinrelating to methods for differentiating embryonic stem cells areincorporated by reference in their entirety herein.

[0129] Thus, using known methods and culture medium, one skilled in theart may culture the subject CICM cells, including genetically engineeredor transgenic CICM cells, to obtain desired differentiated cell types,e.g., neural cells, muscle cells, hematopoietic cells, etc.

[0130] The subject CICM cells may be used to obtain any desireddifferentiated cell type. Therapeutic usages of such differentiatedhuman cells are unparalleled. For example, human hematopoietic stemcells may be used in medical treatments requiring bone marrowtransplantation. Such procedures are used to treat many diseases, e.g.,late stage cancers such as ovarian cancer and leukemia, as well asdiseases that compromise the immune system, such as AIDS. Hematopoieticstem cells can be obtained, e.g., by fusing adult somatic cells of acancer or AIDS patient, e.g., epithelial cells or lymphocytes with anenucleated oocyte, obtaining CICM cells as described above, andculturing such cells under conditions which favor differentiation, untilhematopoietic stem cells are obtained. Such hematopoietic cells may beused in the treatment of diseases including cancer and AIDS.

[0131] Alternatively, adult somatic cells from a patient with aneurological disorder may be fused with an enucleated oocyte, human CICMcells obtained therefrom, and such cells cultured under differentiationconditions to produce neural cell lines. Specific diseases treatable bytransplantation of such human neural cells include, by way of example,Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, amongothers. In the specific case of Parkinson's disease, it has beendemonstrated that transplanted fetal brain neural cells make the properconnections with surrounding cells and produce dopamine. This can resultin long-term reversal of Parkinson's disease symptoms.

[0132] The great advantage of the subject invention is that it providesan essentially limitless supply of isogenic or syngenic human cellssuitable for transplantation. Therefore, it will obviate the significantproblem associated with current transplantation methods, i.e., rejectionof the transplanted tissue which may occur because of host-vs-graft orgraft-vs-host rejection. Conventionally, rejection is prevented orreduced by the administration of anti-rejection drugs such ascyclosporine. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as being veryexpensive. The present invention should eliminate, or at least greatlyreduce, the need for anti-rejection drugs.

[0133] Other diseases and conditions treatable by isogenic cell therapyinclude, by way of example, spinal cord injuries, multiple sclerosis,muscular dystrophy, diabetes, liver diseases, i.e.,hypercholesterolemia, heart diseases, cartilage replacement, burns, footulcers, gastrointestinal diseases, vascular diseases, kidney disease,urinary tract disease, and aging related diseases and conditions.

[0134] This methodology can be used to replace defective genes, e.g.,defective immune system genes, cystic fibrosis genes, or to introducegenes which result in the expression of therapeutically beneficialproteins such as growth factors, lymphokines, cytokines, enzymes, etc.For example, the gene encoding brain derived growth factor may beintroduced into human CICM cells, the cells differentiated into neuralcells and the cells transplanted into a Parkinson's patient to retardthe loss of neural cells during such disease.

[0135] Previously, cell types transfected with BDNF varied from primarycells to immortalized cell lines, either neural or non-neural (myoblastand fibroblast) derived cells. For example, astrocytes have beentransfected with BDNF gene using retroviral vectors, and the cellsgrafted into a rat model of Parkinson's disease (Yoshimoto et al., BrainResearch, 691:25-36, (1995)).

[0136] This ex vivo therapy reduced Parkinson's-like symptoms in therats up to 45% 32 days after transfer. Also, the tyrosine hydroxylasegene has been placed into astrocytes with similar results (Lundberg etal., Develop. Neurol., 139:39-53 (1996) and references cited therein).

[0137] However, such ex vivo systems have problems. In particular,retroviral vectors currently used are down-regulated in vivo and thetransgene is only transiently expressed (review by Mulligan, Science,260:926-932 (1993)). Also, such studies used primary cells, astrocytes,which have finite life span and replicate slowly. Such propertiesadversely affect the rate of transfection and impede selection of stablytransfected cells. Moreover, it is almost impossible to propagate alarge population of gene targeted primary cells to be used in homologousrecombination techniques. By contrast, the difficulties associated withretroviral systems should be eliminated by the use of mammalian CICMcells.

[0138] Genes which may be introduced into the subject CICM cellsinclude, by way of example, epidermal growth factor, basic fibroblastgrowth factor, glial derived neurotrophic growth factor, insulin-likegrowth factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliaryneurotrophic factor, AFT-1, cytokine genes (interleukins, interferons,colony stimulating factors, tumor necrosis factors (alpha and beta),etc.), genes encoding therapeutic enzymes, etc.

[0139] In addition to the use of human CICM cells in cell, tissue andorgan transplantation, the present invention also includes the use ofnon-human cells in the treatment of human diseases. Thus, CICM cells, NTfetuses and NT and chimeric offspring (transgenic or non-transgenic) ofany species may be used in the treatment of human disease conditionswhere cell, tissue or organ transplantation is warranted. In general,CICM cell, fetuses and offspring according to the present invention canbe used within the same species (autologous, syngenic or allografts) oracross species (xenografts). For example, brain cells from bovine NTfetuses may be used to treat Parkinson's disease.

[0140] Also, the subject CICM cells, preferably human cells, may be usedas an in vitro model of differentiation, in particular for the study ofgenes which are involved in the regulation of early development. Also,differentiated cell tissues and organs using the subject CICM cells maybe used in drug studies.

[0141] Further, the subject CICM cells may be used as nuclear donors forthe production of other CICM cells and cell colonies.

[0142] In order to more clearly describe the subject invention, thefollowing examples are provided.

EXAMPLE 1

[0143] Isolation of Primary Cultures of Bovine and Porcine Embryonic andAdult Bovine Fibroblast Cells.

[0144] Primary cultures of bovine and porcine fibroblasts were obtainedfrom fetuses (45 days of pregnancy for cattle and 35 days for pigfetuses). The head, liver, heart and alimentary tract were asepticallyremoved, the fetuses minced and incubated for 30 minutes at 37° C. inprewarmed trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, GrandIsland, N.Y.). Fibroblast cells were plated in tissue culture dishes andcultured in alpha-MEM, medium (Bio Whittaker, Walkersville, Md.)supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, Utah),penicillin (100 IU/ml) and streptomycin (50 μl/ml). The fibroblasts weregrown and maintained in a humidified atmosphere with 5% CO₂ in air at37° C.

[0145] Adult fibroblast cells were isolated from the lung of a cow(approximately five years of age). Minced lung tissue was incubatedovernight at 10° C. in trypsin EDTA solution (0.05% trypsin/0.02% EDTA;GIBCO, Grand Island, N.Y.). The following day tissue and anydisassociated cells were incubated for one hour at 37° C. in prewarmedtrypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island,N.Y.) and processed through three consecutive washes and trypsinincubations (one hr). Fibroblast cells were plated in tissue culturedishes and cultured in alpha-MEM medium (Bio Whittaker, Walkersville,Md.) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen,Utah), penicillin (100 IU/ml) and streptomycin (50 μl/ml). Thefibroblast cells can be isolated at virtually any time in development,ranging from approximately post embryonic disc stage through adult lifeof the animal (bovine day 12 to 15 after fertilization to 10 to 15 yearsof age animals). This procedure can also be used to isolate fibroblastsfrom other mammals, including mice.

[0146] Introduction of a Marker Gene (Foreign Heterologous DNA) intoEmbryonic and Adult Fibroblast Cells.

[0147] The following electroporation procedure was conducted for bothembryonic (cattle and pigs) and adult (cattle) fibroblast cells.Standard microinjection procedures may also be used to introduceheterologous DNA into fibroblast cells, however, in this exampleelectroporation was used because it is an easier procedure.

[0148] Culture plates containing propagating fibroblast cells wereincubated in trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO,Grand Island, N.Y.) until the cells were in a single cell suspension.The cells were spun down at 500× g and re-suspended at 5 million cellsper ml with phosphate buffered saline (PBS).

[0149] The reporter gene construct contained the cytomegaloviruspromoter and the beta-galactosidase, neomycin phosphotransferase fusiongene (beta-GEO). The reporter gene and the cells at 50 μg/ml finalconcentration were added to the electroporation chamber. After theelectroporation pulse, the fibroblast cells were transferred back intothe growth medium (alpha-MEM medium (Bio Whittaker, Walkersville, Md.)supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT),penicillin (100 IU/ml) and streptomycin (50 μl/ml)).

[0150] The day after electroporation, attached fibroblast cells wereselected for stable integration of the reporter gene. G418 (400 μg/ml)was added to growth medium for 15 days (range: 3 days until the end ofthe cultured cells' life span). This drug kills any cells without thebeta-GEO gene, since they do not express the neo resistance gene. At theend of this time, colonies of stable transgenic cells were present. Eachcolony was propagated independently of each other. Transgenic fibroblastcells were stained with X-gal to observe expression ofbeta-galactosidase, and confirmed positive for integration using PCRamplification of the beta-GEO gene and run out on an agarose gel.

[0151] Use of Transgenic Fibroblast Cells in Nuclear Transfer Proceduresto Create CICM Cell Lines and Transgenic Fetuses.

[0152] One line of cells (CL-1) derived from one colony of bovineembryonic fibroblast cells was used as donor nuclei in the nucleartransfer (NT) procedure. General NT procedures are described above.

[0153] Slaughterhouse oocytes were matured in vitro. The oocytes werestripped of cumulus cells and enucleated with a beveled micropipette atapproximately 18 to 20 hrs post maturation (hpm). Enucleation wasconfirmed in TL-HEPES medium plus Hoechst 33342 (3 μg/ml; Sigma).Individual donor cells (fibroblasts) were then placed in theperivitelline space of the recipient oocyte. The bovine oocyte cytoplasmand the donor nucleus (NT unit) were fused together using electrofusiontechniques.

[0154] One fusion pulse consisting of 120 V for 15 μsec in a 500 μm gapchamber was applied to the NT unit. This occurred at 24 hpm. The NTunits were placed in CR1aa medium until 26 to 27 hpm.

[0155] The general procedure used to artificially activate oocytes hasbeen described above. NT unit activation was initiated between 26 and 27hpm. Briefly, NT units were exposed for four min to ionomycin (5 μM;CalBiochem, La Jolla, Calif.) in TL-HEPES supplemented with 1 mg/ml BSAand then washed for five min in TL-HEPES supplemented with 30 mg/ml BSA.Throughout the ionomycin treatment, NT units were also exposed to 2 mMDMAP (Sigma). Following the wash, NT units were then transferred into amicrodrop of CR1aa culture medium containing 2 mM DMAP (Sigma) andcultured at 38.5° C. 5% CO₂ for four to five hrs. The embryos werewashed and then placed in CR1aa medium plus 10% FCS and 6 mg/ml BSA infour well plates containing a confluent feeder layer of mouse embryonicfibroblast. The NT units were cultured for three more days at 38.5° C.and 5% CO₂. Culture medium was changed every three days until days 5 to8 after activation. At this time blastocyst stage NT embryos can be usedto produce transgenic CICM (cultured inner cell mass) cell lines orfetuses. The inner cell mass of these NT units can be isolated andplated on a feeder layer. Also, NT units were transferred into recipientfemales. The pregnancies were aborted at 35 days of gestation. Thisresulted in two cloned transgenic fetuses having the beta-GEO gene inall tissues checked. Thus, this is a fast and easy method of makingtransgenic CICM cell lines and fetuses. This procedure is generallyconducive to gene targeted CICM cell lines and fetuses.

[0156] The table below summarizes the results of these experiments.donor cleavage blastocysts CICM* transgeni cell type n (%) (%) lines (%)(%) CL-1 bovine 412 220 (53) 40 (10%) 22 (55%) embryonic fibroblast(bGEO) CL-1 bovine 505 46 (9%) 4 fetuses embryonic 16 embry fibroblast(bGEO) CICM cell 709 5 (0.7%) line derived from CL-1 NT embryos

EXAMPLE 2

[0157] Chimeric Fetuses Derived from Transgenic CICM Cells. TheTransgenic CICM Cell Line was Derived Originally from a Transgenic NTUnit (Differentiated Cell).

[0158] A CICM line derived from transgenic NT embryos (a CL-1 celltransferred into an enucleated oocyte) was used to produce chimericembryos and fetuses. Colonies of transgenic CICM cells weredisaggregated either using 1-5 mg/ml pronase or 0.05% trypsin/EDTAcombined with mechanical disaggregation methods so that clumps of fiveor fewer cells were produced. Trypsin or pronase activity wasinactivated by passing the cells through multiple washes of 30 to 100%fetal calf serum. The disaggregated cells were placed inmicromanipulation plates containing TL-HEPES medium. Fertilized embryoswere also placed in these plates and micromanipulation tools were usedto produce the chimeric embryos. Eight to ten transgenic CICM cells wereinjected into 8-16 cell stage fertilized embryos. These embryos werecultured in vitro to the blastocyst stage and then transferred intorecipient animals.

[0159] A total of 6 blastocyst stage chimeric embryos werenon-surgically transferred into two recipient females. After five weeksof gestation 3 fetuses were recovered. Several tissues of the threefetuses, including germ cells of the gonad (suggesting germ-linechimeras), were screened by PCR amplification and southern blothybridization of the amplified product to a beta-galactosidase fragment.Of the three fetuses, two were positive for contribution from thetransgenic CICM cells. Both of these fetuses had transgenic CICMcontribution to the gonad.

[0160] Transgenic NT Embryos Derived from Transgenic CICM Cell Lines.The Transgenic CICM Cell Line was Derived Originally from a TransgenicNT Unit (Differentiated Cell).

[0161] The same transgenic CICM cell lines were used to produce NTembryos. The NT procedures described in Example 1 were used except thatCICM cells instead of fibroblast cells were used as the donor cell fusedwith the enucleated oocyte. Colonies of transgenic CICM cells weredisaggregated either using 1-5 mg/ml pronase or 0.05% trypsin/EDTAcombined with mechanical disaggregation methods so that clumps of fiveor fewer cells were produced. Trypsin or pronase activity wasinactivated by passing the cells through multiple washes of 30 to 100%fetal calf serum before transferring the cells into enucleated oocytes.Results are reported in Table 1 (third group). Five blastocyst stageembryos were produced.

What is claimed is:
 1. A method of cloning a mammal, comprising: (i)inserting a desired differentiated mammalian cell or cell nucleus intoan enucleated mammalian oocyte of the same species as the differentiatedcell or cell nucleus, under conditions suitable for the formation of anuclear transfer (NT) unit; (ii) activating the resultant nucleartransfer unit; (iii) culturing said activated nuclear transfer unituntil greater than the 2-cell developmental stage; and (iv) transferringsaid cultured NT unit to a host mammal such that the NT unit developsinto a fetus.
 2. The method according to claim 1, which furthercomprises developing the fetus to an offspring.
 3. The method accordingto claim 1, wherein a desired DNA is inserted, removed or modified insaid differentiated mammalian cell or cell nucleus, thereby resulting inthe production of a genetically altered NT unit.
 4. The method accordingto claim 3, which further comprises developing the fetus to anoffspring.
 5. The method according to claim 1, wherein thedifferentiated mammalian cell or cell nucleus is derived from mesoderm.6. The method according to claim 1, wherein the differentiated mammaliancell or cell nucleus is derived from ectoderm.
 7. The method accordingto claim 1, wherein the differentiated mammalian cell or cell nucleus isderived from endoderm.
 8. The method according to claim 1, wherein thedifferentiated mammalian cell or cell nucleus is a fibroblast cell orcell nucleus.
 9. The method according to claim 1, wherein thedifferentiated mammalian cell or cell nucleus is from an ungulate. 10.The method according to claim 9, wherein the ungulate is selected fromthe group consisting of bovine, ovine, porcine, equine, caprine andbuffalo.
 11. The method according to claim 1, wherein the differentiatedmammalian cell or cell nucleus is an adult cell or cell nucleus.
 12. Themethod according to claim 1, wherein the differentiated mammalian cellor cell nucleus is an embryonic or fetal cell or cell nucleus.
 13. Themethod according to claim 1, wherein the enucleated oocyte is maturedprior to enucleation.
 14. The method according to claim 1, wherein thefused nuclear transfer unit is activated by exposure to ionomycin and6-dimethylaminopurine.
 15. The method according to claim 3, whereinmicroinjection is used to insert a heterologous DNA.
 16. The methodaccording to claim 3, wherein electroporation is used to insert aheterologous DNA.
 17. A fetus obtained according to the method ofclaim
 1. 18. An offspring obtained according to the method of claim 2.19. Progeny of the offspring according to claim
 18. 20. A transgenicfetus obtained according to the method of claim
 3. 21. A transgenicoffspring obtained according to the method of claim
 4. 22. Progeny ofthe offspring according to claim
 21. 23. The method according to claim1, which further comprises combining the cloned NT unit with afertilized embryo to produce a chimeric embryo.
 24. The method accordingto claim 23, which further comprises developing the fetus to anoffspring.
 25. A fetus obtained according to the method of claim
 23. 26.An offspring obtained according to the method of claim
 24. 27. Progenyof the mammal according to claim
 26. 28. A method of producing a CICMcell line, comprising: (i) inserting a desired differentiated mammaliancell or cell nucleus into an enucleated mammalian oocyte of the samespecies as the differentiated cell or cell nucleus, under conditionssuitable for the formation of a nuclear transfer (NT) unit; (ii)activating the resultant nuclear transfer unit; (iii) culturing saidactivated nuclear transfer unit until greater than the 2-celldevelopmental stage; and (iv) culturing cells obtained from saidcultured NT unit to obtain a CICM cell line.
 29. A CICM cell lineobtained according to the method of claim
 28. 30. The method accordingto claim 28, wherein a desired DNA is inserted, removed or modified insaid differentiated mammalian cell or cell nucleus, thereby resulting inthe production of a genetically altered NT unit.
 31. A transgenic CICMcell line obtained according to claim
 30. 32. The method of claim 28,wherein the resultant CICM cell line is induced to differentiate. 33.Differentiated cells obtained by the method of claim
 32. 34. Humandifferentiated cells obtained by the method of claim
 32. 35. A method oftherapy which comprises administering to a patient in need of celltransplantation therapy isogenic differentiated cells according to claim34.
 36. The method of claim 35, wherein said cell transplantationtherapy is effected to treat a disease or condition selected from thegroup consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, ALS, spinal cord defects or injuries, multiplesclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes,heart disease, cartilage defects or injuries, bums, foot ulcers,vascular disease, urinary tract disease, AIDS and cancer.
 37. A methodof therapy which comprises administering to a human patient in need ofcell transplantation therapy xenogenic differentiated cells according toclaim
 33. 38. The method according to claim 37 wherein the xenogenicdifferentiated cells are bovine cells.
 39. The method of claim 37,wherein said cell transplantation therapy is effected to treat a diseaseor condition selected from the group consisting of Parkinson's disease,Huntington's disease, Alzheimer's disease, ALS, spinal cord defects orinjuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liverdisease, diabetes, heart disease, cartilage defects or injuries, burns,foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.40. The method of claim 35, wherein the differentiated human cells arehematopoietic cells or neural cells.
 41. The method of claim 35, whereinthe therapy is for treatment of Parkinson's disease and thedifferentiated cells are neural cells.
 42. The method of claim 35,wherein the therapy is for the treatment of cancer and thedifferentiated cells are hematopoietic cells.
 43. A method of therapywhich comprises administering to a human patient in need of celltransplantation therapy xenogenic cells obtained from a fetus accordingto claim
 17. 44. The method according to claim 43 wherein the xenogeniccells are bovine cells.
 45. The method of claim 43, wherein said celltransplantation therapy is effected to treat a disease or conditionselected from the group consisting of Parkinson's disease, Huntington'sdisease, Alzheimer's disease, ALS, spinal cord defects or injuries,multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease,diabetes, heart disease, cartilage defects or injuries, bums, footulcers, vascular disease, urinary tract disease, AIDS and cancer.
 46. Amethod of therapy which comprises administering to a human patient inneed of cell transplantation therapy xenogenic cells obtained from anoffspring according to claim
 18. 47. The method according to claim 46wherein the xenogenic cells are bovine cells.
 48. The method of claim46, wherein said cell transplantation therapy is effected to treat adisease or condition selected from the group consisting of Parkinson'sdisease, Huntington's disease, Alzheimer's disease, ALS, spinal corddefects or injuries, multiple sclerosis, muscular dystrophy, cysticfibrosis, liver disease, diabetes, heart disease, cartilage defects orinjuries, burns, foot ulcers, vascular disease, urinary tract disease,AIDS and cancer.
 49. A method of therapy which comprises administeringto a human patient in need of cell transplantation therapy xenogenictransgenic cells obtained from a transgenic fetus according to claim 20.50. The method according to claim 49 wherein the xenogenic transgeniccells are bovine cells.
 51. The method of claim 49, wherein said celltransplantation therapy is effected to treat a disease or conditionselected from the group consisting of Parkinson's disease, Huntington'sdisease, Alzheimer's disease, ALS, spinal cord defects or injuries,multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease,diabetes, heart disease, cartilage defects or injuries, bums, footulcers, vascular disease, urinary tract disease, AIDS and cancer.
 52. Amethod of therapy which comprises administering to a human patient inneed of cell transplantation therapy xenogenic transgenic cells obtainedfrom a transgenic offspring according to claim
 21. 53. The methodaccording to claim 52 wherein the xenogenic transgenic cells are bovinecells.
 54. The method of claim 52, wherein said cell transplantationtherapy is effected to treat a disease or condition selected from thegroup consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, ALS, spinal cord defects or injuries, multiplesclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes,heart disease, cartilage defects or injuries, bums, foot ulcers,vascular disease, urinary tract disease, AIDS and cancer.
 55. The methodaccording to claim 28, which further comprises combining the cloned NTunit with a fertilized embryo to produce a chimera.
 56. The methodaccording to claim 55, which further comprises developing the chimericCICM cell line to a chimeric embryo.
 57. A chimeric embryo obtainedaccording to claim
 56. 58. The method according to claim 56, whichfurther comprises developing the chimeric embryo to a chimeric fetus.59. A chimeric fetus obtained according to claim
 58. 60. The methodaccording to claim 58, which further comprises developing the chimericfetus to a chimeric offspring.
 61. A chimeric offspring obtainedaccording to claim
 60. 62. The method according to claim 55, wherein adesired DNA is inserted, removed or modified in said differentiatedmammalian cell or cell nucleus, thereby resulting in the production of agenetically altered NT unit.
 63. The method according to claim 62, whichfurther comprises developing the chimeric CICM cell line to a chimericembryo.
 64. A chimeric embryo obtained according to claim
 63. 65. Themethod according to claim 63, which further comprises developing thechimeric embryo to a chimeric fetus.
 66. A chimeric fetus obtainedaccording to claim
 65. 67. The method according to claim 65, whichfurther comprises developing the chimeric fetus to a chimeric offspring.68. A chimeric offspring obtained according to claim
 67. 69. A method ofcloning a mammal, comprising: (i) inserting a desired differentiatedCICM cell or cell nucleus into an enucleated mammalian oocyte of thesame species as the differentiated CICM cell or cell nucleus, underconditions suitable for the formation of a nuclear transfer (NT) unit;(ii) activating the resultant nuclear transfer unit; (iii) culturingsaid activated nuclear transfer unit until greater than the 2-celldevelopmental stage; and (iv) transferring said cultured NT unit to ahost mammal such that the NT unit develops into a fetus.
 70. The methodaccording to claim 69, which further comprises developing the fetus toan offspring.
 71. A fetus obtained according to the method of claim 69.72. An offspring obtained according to the method of claim
 70. 73. Anorgan for use as an organ xenograft, which is obtained from theoffspring according to claim
 18. 74. An organ for use as an organxenograft, which is obtained from the offspring according to claim 21.75. An organ for use as an organ xenograft, which is obtained from theoffspring according to claim
 26. 76. An organ for use as an organxenograft, which is obtained from the offspring according to claim 68.77. An organ for use as an organ xenograft, which is obtained from theoffspring according to claim 72.